First Report of Soybean Rust Caused by Phakopsora pachyrhizi in Ghana.

Nigeria is the only country in West Africa where soybean rust, caused by Phakopsora pachyrhizi, has been officially reported (1). During a disease survey in Ghana during October 2006, soybean (Glycine max) leaves with rust symptoms (tan, angular lesions with erumpent sori exuding urediniospores) were observed in 11 fields in the following districts: Kassena Nankana in the Upper East Region; East Gonja, Central Gonja, and Tolon-Kumbungu in the Northern Region; and Ejisu-Juabeng in the Ashanti Region. Disease incidence in these fields ranged from 50 to 100% and disease severity ranged between 3 and 40% of the leaf area on infected plants. Urediniospores were hyaline, minutely echinulate, and 23 to 31 × 14 to 18 µm. Within a week of collection, leaf samples were sent to the USDA-ARS Foreign Disease-Weed Science Research Unit for verification of pathogen identity. DNA was extracted from leaf pieces containing sori with the Qiagen DNeasy Plant Mini kit (Valencia, CA), and all 11 field samples amplified in a real-time fluorescent PCR with the P. pachyrhizi-specific primers Ppm1 and Ppa2 (2). Sequence alignment of the internal transcribed spacer (ITS) region 2 further confirmed the identification as P. pachyrhizi (2). Infected leaves from three fields were separately washed in sterile water to collect urediniospores that were used to separately inoculate three detached leaves (for each isolate) of susceptible cultivar TGx 1485-1D (3). The abaxial surface of detached leaves was sprayed with 400 µl of spore suspension (1 × 106 spores per ml). A single leaf piece was placed in a 9-cm-diameter petri dish with adaxial side appressed on 1% technical agar amended with 10 µg/ml of kinetin. Lactic acid (1.5 ml/liter) and benomyl (12.5 mg/liter) were added to the agar medium to inhibit growth of saprophytic fungi and bacteria. Petri dishes were incubated at 20°C with a 12-h light/12-h dark cycle. Lesions on inoculated leaves developed 5 to 6 days after inoculation (DAI), and pustules (105 to 120 µm) formed 7 to 8 DAI and erupted 3 days later exuding columns of urediniospores similar in size to the initially collected isolates. Inoculating another set of detached leaves with a spore suspension (1 × 106 spores per ml) from the first set of detached leaves resulted in typical rust symptoms. The PCR assay, alignment of ITS region 2, morphological characters of the isolates, and pathogenicity tests demonstrate that P. pachyrhizi occurs in Ghana. To our knowledge, this is the first report of P. pachyrhizi in Ghana. References: (1) O. A. Akinsanmi et al. Plant Dis. 85:97, 2001. (2) R. D. Frederick et al. Phytopathology 92:217, 2002. (3) M. Twizeyimana et al. Online publication. http://www.plantmanagementnetwork.org/ infocenter/topic/soybeanrust/2006/posters/41.asp. Plant Management Network, 2006.

First Report of Phakopsora pachyrhizi, Cause of Soybean Rust, on Neonotonia wightii in Paraguay.

Phakopsora pachyrhizi Syd. & P. Syd., the cause of soybean rust, was first observed on soybean (Glycine max (L.) Merr.) in South America in the district of Itapúa in Paraguay during March, 2001 (2). The disease is now widespread in soybean-production areas in South America on soybean and kudzu (Pueraria lobata (Willd.) Ohwi). On 15 March 2006, leaves of the perennial legume Neonotonia wightii (Graham ex Wight & Arn.) Lackey with lesions and rust sori were observed in the Reserva Biológica de Itabó, Departamento Alto Paraná. Lesions were scattered, most contained a single uredinium, mostly hypophyllous, and appeared to be new infections. Lesions with several uredinia, which are indicative of older infections on soybean, were also observed. Sori (Malupa-type) contained hyaline, peripheral, cylindric to clavate paraphyses measuring 24 to 45 × 6 to 13 µm and urediniospores that were hyaline, ovoid to globose, and measuring 20 to 40 × 14 to 25 µm with an echinulate spore wall, characteristics typical of a Phakopsora sp. DNA extracted from sori from leaves of N. wightii was amplified in a real-time fluorescent polymerase chain reaction with the P. pachyrhizi-specific primers Ppm1 and Ppa2 (1). Sequence alignment of the internal transcribed spacer region 2 further confirmed the identification as P. pachyrhizi (1). The host identification was confirmed by J. Kirkbride, USDA/ARS/SBML, using the Smithsonian Institution Department of Botany, U.S. National Herbarium. To our knowledge, this is the first confirmed report of natural infection of P. pachyrhizi on a host other than soybean or kudzu in South America. Voucher specimens were deposited in the herbarium of the Facultad Ciencias Químicas of the Universidad Nacional de Asunción of Paraguay (FCQ) and the National Fungus Collection (Accession No. BPI 875340). References: (1) R. D. Frederick et al. Phytopathology 92:217, 2002. (2) W. Morel and J. Yorinori. Bol. Divulg. No. 44. Ministerio de Agricultura y Ganadería, Centro Regional de Investigación Agrícola, Capitán Miranda, Paraguay, 2002.

First Report of Rust Caused by Phakopsora pachyrhizi on Soybean in Democratic Republic of Congo.

Nigeria (1) and Uganda (3) are the closest countries to the Democratic Republic of Congo (DRC) where soybean rust caused by Phakopsora pachyrhizi has been reported. In February 2007, during a disease survey in DRC, soybean (Glycine max) leaves with rust symptoms (tan, angular lesions with erumpent sori exuding urediniospores) were observed in 10 fields in the following areas in Bas Congo Province: Bangu, Kimpese, Kolo-Fuma, Lukala, Mbanza-Ngungu, Mpalukide, Mvuazi, and Ntemo. Rust incidence in these fields ranged from 85 to 100%, while severity ranged between 3 and 35% of the leaf area on infected plants. Urediniospores were hyaline, minutely echinulate, and 23 to 31 × 16 to 20 µm. Within a week of collection, infected leaf samples were sent to the USDA-ARS Foreign Disease-Weed Science Research Unit (FDWSRU) for pathogen identification. DNA was extracted from sections of leaves containing sori with the Qiagen DNeasy Plant Mini kit (Valencia, CA), and all 10 field samples amplified in a real-time fluorescent PCR with the P. pachyrhizi-specific primers Ppm1 and Ppa2 (2). Infected leaves of cultivar Vuangi collected from one field each in the INERA Research Station, Kimpese-Crawford, and Kimpese-Ceco were separately washed in sterile water to collect urediniospores that were used to separately inoculate three detached leaves of susceptible cultivar TGx 1485-1D (4). Lesions on inoculated leaves developed 5 days after inoculation (DAI), and pustules (110 to 130 µm) formed 7 DAI and erupted 2 days later exuding columns of urediniospores similar in size to the initially collected isolates. Inoculation of another set of detached leaves with a spore suspension (1 × 106 spores per ml) from the first set of detached leaves resulted in typical rust symptoms. Seedlings of cultivar Williams also showed typical rust symptoms when inoculated separately with urediniospores collected from nine fields (i.e., all except Kimpese-Ceco, which was infective in the detached leaf assay). Inoculation and incubation were carried out at the FDWSRU Plant Pathogen Containment Facility at Fort Detrick as described earlier (2). The PCR assay, morphological characters of the isolates, and pathogenicity tests demonstrate that P. pachyrhizi occurs in DRC. To our knowledge, this is the first report of P. pachyrhizi infecting soybean in DRC. References: (1) O. A. Akinsanmi et al. Plant Dis. 85:97, 2001. (2) R. D. Frederick et al. Phytopathology 92:217, 2002. (3) E. Kawuki et al. J. Phytopathol. 151:7, 2003. (4) M. Twizeyimana et al. Online publication. http://www.plantmanagementnetwork.org/ infocenter/topic/soybeanrust/2006/posters/41.asp. Plant Management Network, 2006.

Evaluation of Virulence of Phakopsora pachyrhizi and P. meibomiae Isolates.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi and recently discovered for the first time in continental United States, has been of concern to the U.S. agricultural industry for more than 30 years. Since little soybean rust resistance is known, and resistance is often difficult to detect or quantitate, we initiated a project to develop a better, more quantitative, method. The methodology determined the average numbers and diameters of uredinia in lesions that developed on leaves of inoculated plants 14 days after inoculation. It was used to compare virulence of P. pachyrhizi isolates from Asia and Australia and P. meibomiae from Puerto Rico and Brazil, collected as many as 30 years earlier, with isolates of P. pachyrhizi recently collected from Africa or South America. Susceptible reactions to P. pachyrhizi resulted in tan-colored lesions containing 1 to 14 uredinia varying greatly in size within individual lesions. In contrast, on these same genotypes at the same time of year, resistance to other P. pachyrhizi isolates was typified by 0 to 6 small uredinia in reddish-brown to dark-brown lesions. Using appropriate rust resistant and rust susceptible genotypes as standards, examination of uredinia 14 days after inoculation allowed quantitative comparisons of sporulation capacities, one measure of susceptibility or resistance to soybean rust. The study verified the presence and ability to detect all known major genes for resistance to soybean rust in the original sources of resistance. It demonstrated that soybean lines derived from the original PI sources, and presumed to possess the resistance genes, in actuality may lack the gene or express an intermediate reaction to the rust pathogen. We suggest that a determination of numbers and sizes of uredinia will detect both major gene and partial resistance to soybean rust.

Building academic and practical knowledge in nursing through TB skin testing certification in a BSN curriculum.

Tuberculosis (TB) is a communicable disease with worldwide health care consequences. After decades of declining incidence there was a 14% increase in the United States from 1985 to 1992, although rates are now declining due to focused programs. The State of Indiana mandates continuous TB monitoring by health care workers who have completed an educational course. The Indiana State Department of Health (ISDH) and the American Lung Association designed an educational program called a Basic TB Skin Test Course, which is used by the Community Health Nursing faculty at Indiana University School of Nursing (IUSON) in the baccalaureate curriculum to assist students in meeting state requirements. All students receive the didactic TB content of the course in the epidemiological concepts unit. Students can elect to receive certification by attending an additional 2-hour clinical session. Over 500 community health nursing students have completed the Basic TB Skin Test course in the past 5 years. The outcomes are positive for students, community, and public health.

Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase.

Formaldehyde, a major industrial chemical, is classified as a carcinogen because of its high reactivity with DNA. It is inactivated by oxidative metabolism to formate in humans by glutathione-dependent formaldehyde dehydrogenase. This NAD(+)-dependent enzyme belongs to the family of zinc-dependent alcohol dehydrogenases with 40 kDa subunits and is also called ADH3 or chi-ADH. The first step in the reaction involves the nonenzymatic formation of the S-(hydroxymethyl)glutathione adduct from formaldehyde and glutathione. When formaldehyde concentrations exceed that of glutathione, nonoxidizable adducts can be formed in vitro. The S-(hydroxymethyl)glutathione adduct will be predominant in vivo, since circulating glutathione concentrations are reported to be 50 times that of formaldehyde in humans. Initial velocity, product inhibition, dead-end inhibition, and equilibrium binding studies indicate that the catalytic mechanism for oxidation of S-(hydroxymethyl)glutathione and 12-hydroxydodecanoic acid (12-HDDA) with NAD(+) is random bi-bi. Formation of an E.NADH.12-HDDA abortive complex was evident from equilibrium binding studies, but no substrate inhibition was seen with 12-HDDA. 12-Oxododecanoic acid (12-ODDA) exhibited substrate inhibition, which is consistent with a preferred pathway for substrate addition in the reductive reaction and formation of an abortive E.NAD(+).12-ODDA complex. The random mechanism is consistent with the published three-dimensional structure of the formaldehyde dehydrogenase.NAD(+) complex, which exhibits a unique semi-open coenzyme-catalytic domain conformation where substrates can bind or dissociate in any order.

Characterization of human alcohol dehydrogenase isoenzymes by capillary isoelectric focusing-mass spectrometry.

The human liver alcohol dehydrogenase (ADH) isoenzymes are currently believed to play a major role in ethanol metabolism, accounting for most of the ethanol oxidized in the liver. They have similar molecular masses and similar isoelectric point (pI) values (the 13 possible isoenzymes having pIs in the range of 8.26-8.87), making their characterization a significant analytical challenge. Capillary isoelectric focusing (CIEF) coupled on-line with electrospray ionization - Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry was applied to separate and characterize mixtures of alphaalpha, beta1beta1 and beta3beta3 ADH isoenzymes. Seven different species were resolved by the separation in the pI 8.26-8.67 range. ESI-FTICR analysis of native ADHs revealed that each noncovalent ADH complex contains two monomeric protein units and four zinc atoms. The combination of CIEF separations with mass spectrometry appears well-suited for detailed characterization of ADH isozymes, and the attomole level sensitivity of FTICR should allow very small samples to be addressed.

The pH-dependent binding of NADH and subsequent enzyme isomerization of human liver beta 3 beta 3 alcohol dehydrogenase.

Human class I beta 3 beta 3 is one of the alcohol dehydrogenase dimers that catalyzes the reversible oxidation of ethanol. The beta 3 subunit has a Cys substitution for Arg-369 (beta 369C) in the coenzyme-binding site of the beta1 subunit. Kinetic studies have demonstrated that this natural mutation in the coenzyme-binding site decreases affinity for NAD+ and NADH. Structural studies suggest that the enzyme isomerizes from an open to closed form with coenzyme binding. However, the extent to which this isomerization limits catalysis is not known. In this study, stopped-flow kinetics were used from pH 6 to 9 with recombinant beta 369C to evaluate rate-limiting steps in coenzyme association and catalysis. Association rates of NADH approached an apparent zero-order rate with increasing NADH concentrations at pH 7.5 (42 +/- 1 s-1). This observation is consistent with an NADH-induced isomerization of the enzyme from an open to closed conformation. The pH dependence of apparent zero-order rate constants fit best a model in which a single ionization limits diminishing rates (pKa = 7.2 +/- 0.1), and coincided with Vmax values for acetaldehyde reduction. This indicates that NADH-induced isomerization to a closed conformation may be rate-limiting for acetaldehyde reduction. The pH dependence of equilibrium NADH-binding constants fits best a model in which a single ionization leads to a loss in NADH affinity (pKa = 8.1 +/- 0. 2). Rate constants for isomerization from a closed to open conformation were also calculated, and these values coincided with Vmax for ethanol oxidation above pH 7.5. This suggests that NADH-induced isomerization of beta 369C from a closed to open conformation is rate-limiting for ethanol oxidation above pH 7.5.

Evaluation of a personal air sampler for twenty-four hour collection of fine particles and semivolatile organics.

The U.S. EPA has conducted an evaluation of a commercially available lightweight fine particle personal sampler for use in human exposure and biomarker studies involving 24-h collections of particulate matter, particle-bound organics such as polycyclic aromatic hydrocarbons (PAHs), and semivolatile organics (PAHs). Independent laboratory evaluation of the prototype design, intended to produce a 2.5-micron aerodynamic diameter cut-point at a flow between 1.5 and 1.7 lpm (liters per minute), indicated that at a challenge flow rate of 1.5 lpm, the sampler provided an aerodynamic cut-point (dp50) of only 1.7 microns. The variance between expected size selection resulted from the prototype's jet diameter dimension being inadvertently based upon a flow rate of 2.0 lpm rather than an intended 1.5-1.7 lpm. Other aerodynamic factors not presently accounted for may also have played an influence. Extrapolated cut-points for flow rates at 1 and 3 lpm for the prototype were determined to be 2.1 and 1.2 microns, respectively. Total losses attributed to unwanted particle retention within the sampler ranged from 10% for 0.91 micron size particles to essentially zero approaching diameters greater than 2.0 microns. The ambient concentration of particles (< 1.7 microns) needed for acceptable gravimetric filter measurements involving 24-h sample collection was determined to be 10 micrograms/m3. Investigation of the sampler to retain and recover PAHs using XAD-2 resin, often of importance in human exposure biomarker studies, indicated that corrected recoveries between 94% and 108% could be obtained for 16 priority pollutant PAH species. Overall evaluation of the personal monitor indicates that it has research utility due to its modular features and size but reconfiguration should be performed that would permit true PM2.5 size selection. The current configuration collects particles less than 2.5 microns at approximately 95% collection efficiency.

cDNA sequence and catalytic properties of a chick embryo alcohol dehydrogenase that oxidizes retinol and 3beta,5alpha-hydroxysteroids.

This study was undertaken to identify the cytosolic 40-kDa zinc-containing alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues. Degenerate oligonucleotide primers were used to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart. cDNA fragments that encode an unknown putative alcohol dehydrogenase as well as the class III alcohol dehydrogenase were identified. The new cDNA hybridized with two messages of approximately 2 and 3 kilobase pairs in the adult chicken liver but not in the adult heart, muscle, testis, or brain. The corresponding complete cDNA clones with a total length of 1390 base pairs were isolated from a chicken liver lambdagt11 cDNA library. The open reading frame encoded a 375-amino acid polypeptide that exhibited 67 and 68% sequence identity with chicken class I and III alcohol dehydrogenases, respectively, and had lower identity with mammalian class II (55-58%) and IV (62%) isozymes. Expression of the new cDNA in Escherichia coli yielded an active alcohol dehydrogenase (ADH-F) with subunit molecular mass of approximately 40 kDa. The specific activity of the recombinant enzyme, calculated from active site titration of NADH binding, was 3.4 min-1 for ethanol at pH 7.4 and 25 degrees C. ADH-F was stereospecific for the 3beta,5alpha- versus 3beta,5beta-hydroxysteroids. The Km value for ethanol at pH 7.4 was 17 mM compared with 56 microM for all-trans-retinol and 31 microM for epiandrosterone. Antiserum against ADH-F recognized corresponding protein in the chicken liver homogenate. We suggest that ADH-F represents a new class of alcohol dehydrogenase, class VII, based on its primary structure and catalytic properties.

X-ray structure of human beta3beta3 alcohol dehydrogenase. The contribution of ionic interactions to coenzyme binding.

The three-dimensional structure of the human beta3beta3 dimeric alcohol dehydrogenase (beta3) was determined to 2.4-A resolution. beta3 was crystallized as a ternary complex with the coenzyme NAD+ and the competitive inhibitor 4-iodopyrazole. beta3 is a polymorphic variant at ADH2 that differs from beta1 by a single amino acid substitution of Arg-369 --> Cys. The available x-ray structures of mammalian alcohol dehydrogenases show that the side chain of Arg-369 forms an ion pair with the NAD(H) pyrophosphate to stabilize the E.NAD(H) complex. The Cys-369 side chain of beta3 cannot form this interaction. The three-dimensional structures of beta3 and beta1 are virtually identical, with the exception that Cys-369 and two water molecules in beta3 occupy the position of Arg-369 in beta1. The two waters occupy the same positions as two guanidino nitrogens of Arg-369. Hence, the number of hydrogen bonding interactions between the enzyme and NAD(H) are the same for both isoenzymes. However, beta3 differs from beta1 by the loss of the electrostatic interaction between the NAD(H) pyrophosphate and the Arg-369 side chain. The equilibrium dissociation constants of beta3 for NAD+ and NADH are 350-fold and 4000-fold higher, respectively, than those for beta1. These changes correspond to binding free energy differences of 3.5 kcal/mol for NAD+ and 4.9 kcal/mol for NADH. Thus, the Arg-369 --> Cys substitution of beta3 isoenzyme destabilizes the interaction between coenzyme and beta3 alcohol dehydrogenase.

Cimetidine inhibition of human gastric and liver alcohol dehydrogenase isoenzymes: identification of inhibitor complexes by kinetics and molecular modeling.

Cimetidine, an H2-receptor antagonist, is one of the most commonly prescribed drugs in the world. It has been reported to increase blood alcohol concentrations in drinking individuals. To determine if this increase could be due to inhibition of alcohol dehydrogenase activity, the effect of the drug on ethanol oxidation by gastric sigma sigma alcohol dehydrogenase and liver beta 2 beta 2, pi pi, and chi chi alcohol dehydrogenase isoenzymes was observed. Cimetidine inhibited all isoenzymes studied except chi chi; the chi chi isoenzyme showed no inhibition up to 5 mM cimetidine. Inhibition of the alcohol dehydrogenase isoenzymes by the H2-receptor antagonists nizatidine, ranitidine, and famotidine was negligible. Docking simulations with the beta 2.NAD+.4-iodopyrazole X-ray structure indicated that cimetidine fit well into the substrate binding site. The substitution on the thiazole ring of nizatidine, however, prevented docking into the binding site. Cimetidine inhibition of ethanol oxidation by sigma sigma and beta 2 beta 2 was competitive with varied ethanol, exhibiting Ki values of 2.8 +/- 0.4 mM and 0.77 +/- 0.07 mM, respectively. Cimetidine inhibition of ethanol oxidation by pi pi was noncompetitive with varied ethanol (Ki = 0.50 +/- 0.03 mM). Inhibition of ethanol oxidation by sigma sigma and beta 2 beta 2 with varied NAD+ was competitive. These results, together with the cimetidine inhibition kinetics of acetaldehyde reduction by sigma sigma and beta 2 beta 2, with either varied NADH or varied acetaldehyde, are consistent with cimetidine binding to two enzyme species. These species are free enzyme and the productive enzyme.NAD+ complex.

Expression and kinetic characterization of recombinant human stomach alcohol dehydrogenase. Active-site amino acid sequence explains substrate specificity compared with liver isozymes.

A full-length 1966-base pair clone of the human class IV alcohol dehydrogenase (sigma-ADH) was isolated from a human stomach cDNA library. The 373-amino acid sigma-ADH encoded by this cDNA was expressed in Escherichia coli. The specific activity of the recombinant enzyme for ethanol oxidation at pH 7.5 and 25 degrees C, calculated from active-site titration of NADH binding, was 92 +/- 9 units/mg. Kinetic analysis of the catalytic efficiency (kcat/KM) of recombinant sigma-ADH for oxidation of primary alcohols indicated broad substrate specificity. Recombinant human sigma-ADH exhibited high catalytic efficiency for oxidation of all-trans-retinol to all-trans-retinal. This pathway is important in the synthesis of the transcriptional regulator all-trans-retinoic acid. Secondary alcohols and 3 beta-hydroxysteroids were inactive with sigma-ADH or were oxidized with very low efficiency. The KM of sigma-ADH for ethanol was 25 mM, and the KM for primary straight chain alcohols decreased substantially as chain length increased. There are important amino acid differences in the alcohol-binding site between the human class IV (sigma) and human class I (beta) alcohol dehydrogenases that appear to explain the high catalytic efficiency for all-trans-retinol, the high kcat for ethanol, and the low catalytic efficiency for secondary alcohols of sigma-ADH relative to beta 1-ADH. For example, modeling the binding of all-trans-retinol in the human beta 1-ADH structure suggested that coordination of retinol to the active-site zinc is hindered by a loop from residues 114 to 120 that is at the entrance to the alcohol-binding site. The deletion of Gly-117 in human sigma-ADH and a substitution of Leu for the bulky Tyr-110 appear to facilitate retinol access to the active-site zinc.

Purification and characterization of a human liver cocaine carboxylesterase that catalyzes the production of benzoylecgonine and the formation of cocaethylene from alcohol and cocaine.

The psychomotor stimulant cocaine is inactivated primarily by hydrolysis to benzoylecgonine, the major urinary metabolite of the drug. A non-specific carboxylesterase was purified from human liver that catalyzes the hydrolysis of the methyl ester group of cocaine to form benzoylecgonine. In the presence of ethanol, the enzyme also catalyzes the transesterification of cocaine producing the pharmacologically active metabolite cocaethylene (benzoylecgonine ethyl ester). The carboxylesterase obeys simple Michaelis-Menten kinetics with Km values of 116 microM for cocaine and 43 mM for ethanol. The enzymatic activity suggests that it may play an important role in regulating the detoxication of cocaine and in the formation of the active metabolite cocaethylene. Additionally, the enzyme catalyzes the formation of ethyloleate from oleic acid and ethanol. The carboxylesterase was purified from autopsy liver by gel filtration, chromatofocusing, ion-exchange, and hydrophobic interaction chromatography to purity by SDS-PAGE and agarose gel isoelectric focusing. The subunit molecular weight was determined to be 59,000 and the native molecular weight was estimated to be 170,000 from a calibrated gel filtration column, suggesting that the active enzyme is a trimer. The isoelectric point was approximately 5.8. Digestion of carbohydrate residues on the protein with an acetylglucosaminidase plus binding to several lectins indicates that the enzyme is glycosylated. The esterase was cleaved with two proteases, and the amino acid sequences from fourteen peptides were used to search GenBank. Two identical matches were found corresponding to carboxylesterase cDNAs from human liver and lung.

Structures of three human beta alcohol dehydrogenase variants. Correlations with their functional differences.

The three-dimensional structures of three variants of human beta alcohol dehydrogenase have been determined to 2.5 A resolution. These three structures differ only in the amino acid at position 47 and the molecules occupying the alcohol binding site. Human beta 1 alcohol dehydrogenase has an Arg at position 47 and was crystallized in a complex with NAD(H) and cyclohexanol. A naturally occurring variant of beta 1 alcohol dehydrogenase, found in approximately 50% of the Asian population, possesses a His at position 47 (beta 2 or beta 47H) and was crystallized in a complex with NAD+ and the inhibitor 4-iodopyrazole. A site-directed mutant of beta 1 alcohol dehydrogenase in which a Gly is substituted for Arg47 (beta 47G) was crystallized in a complex with NAD+. By comparing both the common and unique features of these structures, it is clear that position 47 contributes significantly to the strength of protein-coenzyme interactions. The substitution of Arg47 by His produces an enzyme with a 100-fold lower affinity for coenzyme, but creates no large changes in the enzyme structure. The substitution of Arg47 by Gly produces an enzyme with coenzyme binding characteristics more similar to the wild-type enzyme than to the enzyme with His at position 47, but the structure of the Gly47 variant exhibits differences in and around the coenzyme binding site. These changes involve a rigid-body rotation of the catalytic domain towards the coenzyme domain by approximately 0.8 degrees and local rearrangements of amino acid side-chains, such as a 1.0 A movement of Lys228, relative to the beta 1 enzyme. These structural alterations may compensate for the loss of coenzyme interactions contributed by Arg47 and can explain the high affinity of the Gly47 variant for coenzyme.

Catalytic efficiency of human alcohol dehydrogenases for retinol oxidation and retinal reduction.

Mammalian alcohol dehydrogenase (ADH) is thought to be involved in the reversible oxidation of vitamin A or retinol to retinal for retinoic acid synthesis. Retinoic acid is a potent transcriptional regulator and a morphogen. It was proposed that the competition of consumed ethanol with retinol oxidation by ADH might explain developmental disorders seen with fetal alcohol syndrome. We report herein the relative efficiency (V/Km) of eight human ADH isoenzymes for oxidation of all-trans-retinol and reduction of three retinal isomers (all-trans, 9-cis, and 13-cis-retinal). Class IV sigma sigma and class II pi pi isoenzymes are the most efficient forms, with V/Km values approximately 100 and 30 times greater, respectively, than class I beta 1 beta 1 or gamma 1 gamma 1, sigma sigma exhibits the highest V/Km (1-2 microns-1min-1), followed by pi pi, with V/Km of 0.5-0.6 microns-1min-1 for all-trans-retinol, all-trans-retinal, and 9-cis-retinal. pi pi also has the lowest Km (11-14 microns) for all-trans-retinol and three retinal isomers. alpha alpha shows an intermediate efficiency, with V/Km of 0.09-0.2 microns-1min-1 and a relatively low Km of 16-24 microns for all four substrates. alpha alpha has the highest efficiency of all tested isoenzymes for 13-cis-retinal. Class III chi chi is inactive with all the tested retinoids.(ABSTRACT TRUNCATED AT 250 WORDS)